Four hundred mesh carbon-coated grids (Quantifoil Micro Tools, Jena, Germany) were freshly glow discharged in a PELCO easiGlow system (Ted Pella, Redding, USA) for 30 s at 25 mA. Immediately afterward, 4 μl of an sEV sample was placed on the grid and left for 1 min. Subsequently, excess liquid was removed using filter paper and the grid washed twice with MilliQ water (Merck, Schaffhausen, Switzerland). Next, the grids were negatively stained using two consecutive drops of 1% uranyl acetate for 1.5 min each. After thorough air drying, the grid was imaged using a CCD KeeneView camera within a Morgagni 268 TEM (Thermo Fisher Scientific, Reinach, Switzerland) operating in bright-field mode at 100 kV.
Carbon coated grids
Manufactured by Quantifoil
Sourced in Germany
Carbon coated grids are thin, circular substrates used in electron microscopy. They provide a stable and conductive support for samples to be analyzed under high-vacuum conditions.
Automatically generated - may contain errors
Lab products found in correlation
Morgagni 268 tem, by Thermo Fisher Scientific (3 mentions)
Milli q water, by Merck Group (2 mentions)
Pelco easiglow system, by Ted Pella (2 mentions)
Keeneview camera, by Thermo Fisher Scientific (2 mentions)
Orius 1 k camera, by Ametek (1 mentions)
Cm12 microscope, by Philips (1 mentions)
Jem 2200fs electron microscope, by JEOL (1 mentions)
Gibco fetal bovine serum, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
8 protocols using carbon coated grids
1
Negative Staining of Small Extracellular Vesicles
2
Cryo-EM Grid Preparation and Imaging
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Carbon coated grids (Quantifoil, Hatfield, PA, USA) were glow-discharged using an EMS GlowQube instrument. The samples were then applied to the grids at 0.02–0.04 mg/mL, incubated for 1 min, and blotted. The grids were then washed and stained sequentially with PBS and 2% uranyl acetate and finally air-dried. The micrographs were acquired using a G2 Sphera microscope (Thermofisher, Waltham, MA, USA) operating at 120 kV with a defocus range of −1 to −2 µm. Images were analyzed with Fiji software (ImageJ, 1.53c plus).
3
Negative Staining of Small Extracellular Vesicles
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Four hundred mesh carbon-coated grids (Quantifoil Micro Tools, Jena, Germany) were freshly glow discharged in a PELCO easiGlow system (Ted Pella, Redding, USA) for 30 s at 25 mA. Immediately afterward, 4 μl of an sEV sample was placed on the grid and left for 1 min. Subsequently, excess liquid was removed using filter paper and the grid washed twice with MilliQ water (Merck, Schaffhausen, Switzerland). Next, the grids were negatively stained using two consecutive drops of 1% uranyl acetate for 1.5 min each. After thorough air drying, the grid was imaged using a CCD KeeneView camera within a Morgagni 268 TEM (Thermo Fisher Scientific, Reinach, Switzerland) operating in bright-field mode at 100 kV.
4
Negative Staining Electron Microscopy of Pol I PIC
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The negatively stained samples were prepared on carbon coated grids (Quantifoil). Grids were glow discharged for 10 s, and then 3.5 μl of the sample (0.025 mg/ml) was deposited on the grids and incubated for 1 min. Grids were sequentially washed and stained with 1% (w/v) uranyl acetate solution and air‐dried. Data of Pol I PIC (+DNA) were acquired with Tecnai Polara operating at 100 keV and magnification of 78,000 (1.9 Å pixel size), and data of Pol I PIC (−DNA) were acquired with Tecnai T12 operating at 120 keV and magnification of 68,000 (1.6 Å pixel size). The images were acquired in a defocus range of −1.5 to −2.0 μm and electron dose of 20 e−/Å2 using a 4k × 4k CCD Ultrascan camera.
5
Imaging Bacteriophages via TEM
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To obtain transmission electron microscopy (TEM) images of the bacteriophages, dilutions of the samples were spotted on carbon coated grids (Quantifoil, Großlöbichau, Germany) after glow-discharge, and negatively stained with 2% uranyl acetate. A Philips CM12 microscope was used at 120 kV acceleration voltage. Images were produced using a Gatan Orius 1 k camera.
6
Negative Stain Electron Microscopy for trCLN3-L4 Nanoconstructs
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The size and structure of the trCLN3-L4 nanoconstructs were analyzed by negative stain electron microscopy. Samples were prepared using negative staining74 (link). In brief, carbon coated grids (Quantifoil Micro Tools GmbH, Jena, Germany, 200 mesh) were glow discharged to render the surface hydrophilic prior to applying samples. 10 µL of an aqueous solution of trCLN3-L4 were applied to the grid. Afterward excess solution was carefully blotted off using filter paper followed by three times washing with ddH2O. In the final step, grids were stained with negative staining reagent by placing them (plastic side down) on a 10 µL drop of freshly prepared 2% (v/v) uranyl formiate aqueous staining solution. TEM micrographs were recorded using a JEOL JEM 2200 FS electron microscope (JEOL, Japan) operated at 200 kV. The size of the micelles measured on the TEM images could typically be observed in a range between 20 and 25 nm.
7
TEM Imaging of Extracellular Vesicles
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For transmission electron microscope (TEM) imaging, DyLight −594 labelled EV‐samples were purified as described above. Five microlitre of sample was placed on glow discharged (Pelco, Ted Pella, Redding, CA) carbon‐coated grids (Quantifoil, Grosslöbichau, Germany) and incubated for 3 min. After this interval, excess fluid was removed with filter paper and the grid was washed twice. The wet sample was stained in a drop of 2% uranyl acetate for 1 s followed by a second step for 15 s. Excess moisture was drained with filter paper, and the imaging of the air‐dried grids was done in a TEM Morgagni 268 (Thermo Fisher Scientific) operated at 100 kV acceleration voltage. Data were acquired with a KeenView CCD‐camera (Soft Imaging System, Muenster, Germany; 1376 × 1032 Pixel). In addition, EV samples were imaged using a Tecnai F20 field emission gun (FEG) microscope (Field Electron and Ion Company, Hillsboro, OR), operated at a 120 kV acceleration voltage, and equipped with a combination of CCD (ORIUS SC200 2 K) and direct electron detector (Falcon II 4 K, Thermo Fisher Scientific).
8
Site-Directed Mutagenesis Protocol
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A QuikChange II Site-Directed Mutagenesis Kit, Lipofectamine 2000, Dulbecco’s modified Eagle medium (DMEM) and GIBCO fetal bovine serum were obtained from Invitrogen. Carbon-coated grids were obtained from Quantifoil. Mouse anti-M1 monoclonal antibody and rabbit anti-NP or anti-HA polyclonal antibody were prepared by standard procedures. The origin of other materials was described in the previous study (Zhang et al., 2012 (link)).
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